A microscope operating according to the SPIM method is described in DE 102 57 423 A1. In this microscope, a sample is illuminated by a thin light strip, while observation takes place perpendicularly to the plane of the illuminating light strip. Here, illumination and detection are carried out via two separate optical paths each having separate optics, in particular having two separate objectives perpendicular to each other. The light strip is produced by the illumination objective and a cylindrical optic placed in front of it. For image acquisition, the sample is moved through the light strip, which is stationary relative to the detector, to capture fluorescent and/or scattered light layer by layer using an area detector. The layered-image data thereby obtained can then be assembled into a data set corresponding to a three-dimensional image of the sample. In order to produce as thin a light strip as possible, the illumination objective must have a correspondingly high numerical aperture, and the free working distance of the illumination objective must be correspondingly large in order to prevent collision with the observation objective. This type of perpendicular arrangement of the two objectives can be disadvantageous for imaging certain samples, especially biological ones. For example, it is often not possible to place spherical objects in a collision-free manner under a right-angled objective arrangement. In addition to the extreme requirements in terms of sample preparation, unwanted shading also often occurs in the sample.
In a modified SPIM technique described in WO 2010012980 A1, illumination and detection are performed using the same objective. To this end, the entrance pupil of the objective is decentrally under-illuminated, i.e., the illumination beam passes through a portion of the entrance pupil that is offset transversely from the optical axis. A cylindrical lens arranged in front of the objective produces a light sheet in the sample, which light sheet is oblique with respect to the optical axis of the objective. The sample region illuminated by this light sheet is then in turn imaged by the objective onto a detector. However, this device is designed exclusively for oblique illumination of the sample by means of a light sheet and does not allow for any use deviating therefrom, and especially not for point-by-point confocal scanning of the sample or variation of the spatial light intensity distribution of the light sheet, and in particular, not for illumination by a light strip oriented perpendicular to the optical axis of the objective.
DE 10 2004 034 957 A1 describes an arrangement for microscopic observation of a sample through a microscope objective, in whose housing light guides for the light illuminating the sample are provided outside the lens optic. The illumination light initially proceeds parallel to the optical axis of the objective within the light guide, and then strikes small-aperture reflectors that are mounted on the objective housing and that, with the aid of additional imaging elements, focus the illuminating light into the sample perpendicularly to the optical axis of the microscope objective, and thus perpendicularly to the observation direction. Here too, illumination of the sample occurs in planar fashion according to the SPIM principle. Although the use of a microscope objective configured in this manner does eliminate the need to use an additional objective for the illuminating light, the special design of this special objective with additional light guides and reflectors is technically very complex, and expensive.
With the apparatus known from DE 10 2004 034 957 A1, the problem exists that only objects which fit within the maximum image field of the objective, between the oppositely located reflectors that deflect the illumination light onto the object, can be investigated. A large image field is available, however, only at low magnifications. High-magnification objectives, which as a general rule have a high numerical aperture, are not usable because the sample is larger than the maximum image field, and consequently does not fit between the oppositely located mirror surfaces. Objectives having a low aperture disadvantageously allow the formation of only a relatively thick light strip.